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Abstract:

The invention relates to methods for directing differentiation of stem
cells comprising graphene. In additional embodiments, the invention
relates to methods for repairing and improving bone tissue functions
comprising accelerating differentiation in stem cell growth by exposing
stem cells to graphene and transplanting the graphene with the exposed
stem cells in the tissue at the site of repair.

Claims:

1. A method of directing stem cell differentiation in the absence of
growth factors or external stimulation, comprising: placing stem cells on
a graphene substrate and exposing to a culture media for a period of time
sufficient to allow the stem cells to differentiate in cells of interest
in the absence of growth factors or external stimulation.

2. The method of claim 1, where graphene is single layer graphene.

3. The method of claim 1, where the graphene is multi layer graphene.

4. The method of claim 1, where the graphene is two-dimensional graphene.

5. The method of claim 1, where the graphene is three-dimensional
graphene.

6. The method of claim 1, where the stem cell is a mesenchymal stem cell.

7. The method of claim 1, where the stem cell is a progenitor cell.

8. The method of claim 1, where the graphene is in the culture media.

9. The method of claim 1, where the culture media is stem cell media.

10. The method of claim 1, where the culture media is osteogenic media.

11. The method of claim 1, where the accelerated differentiation is
osteogenic differentiation.

12. The method of claim 1, where the differentiated cell is an
osteoblast.

13. The method of claim 1, where the supporting substrate is a
biocompatible material.

14. A method of repairing and improving bone tissue function comprising
directing stem cell differentiation by placing the stem cells on graphene
in the absence of growth factors or external stimulation and
transplanting the differentiated stem cells in the tissue at the site of
repair.

15. The method of claim 14, where the stem cell is a mesenchymal stem
cell.

16. The method of claim 14, where the culture media is osteogenic media.

17. A composition for accelerating differentiation of stem cells
comprising graphene on an implantable, biocompatible scaffold for support
of tissue growth.

18. The composition of claim 17, where the stem cell is a mesenchymal
stem cell.

19. The composition of claim 17, where the culture media is osteogenic
media.

20. (canceled)

Description:

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application
No. 61/362,506, filed Jul. 8, 2010, which is incorporated herein by
reference in its entirety.

[0003] Therefore, there remains a significant need for development of more
biocompatible scaffolds that allow for better scalability of the
biocompatible scaffold materials and compatibility with implants.

SUMMARY OF THE INVENTION

[0004] In a first main aspect, the invention relates to a method of
directing stem cell differentiation in the absence of growth factors or
external stimulation, comprising: placing stem cells on a graphene
substrate and exposing to a culture media for a period of time sufficient
to allow the stem cells to differentiate in cells of interest in the
absence of growth factors or external stimulation. The graphene can be
single layer, multi-layer, two dimensional or three dimensional. In one
embodiment, the culture media is an osteogenic medium.

[0005] In one embodiment, the stem cells are mesenchymal stem cells. In
another embodiment, the stem cells are progenitor cells.

[0006] In another aspect, the invention relates to a method of repairing
and improving bone tissue function comprising directing stem cell
differentiation by placing the stem cells on graphene, e.g., single layer
or multi-layer, in the absence of growth factors or external stimulation
and transplanting the graphene with the stem cells in the tissue at the
site of repair.

[0007] In a further aspect, the invention relates to a composition for
accelerating differentiation of human mesenchymal stem cells comprising
single layer graphene on an implantable, biocompatible scaffold for
support of tissue growth.

[0008] In another aspect, the invention relates to the use of graphene as
a substrate for stem cell differentiation.

[0009] The present invention provides graphene as a low cost,
biocompatible scaffold that does not hamper the proliferation of human
stem cells and accelerates their specific differentiation into various
cell types.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing will be apparent from the following more particular
description of example embodiments of the invention, as illustrated in
the accompanying drawings in which like reference characters refer to the
same parts, throughout the different views. The drawings are not
necessarily to scale, emphasis instead being placed upon illustrating
embodiments of the present invention.

[0011] FIG. 1 (a) is a graph of cell viability of hMSCs grown on different
substrates including silicon wafer with 300 nm SiO2 (Si/SiO2),
polyethylene terephthalate (PET), and polydimethylsiloxane (PDMS) in
percentage normalized to cover slips used as a reference. FIG. 1 (b-i)
and inset show cell morphology of hMSCs grown on standard cover slips and
on glass slide, Si/SiO2, PET and PDMS with or without graphene.
Scale bars are 100 μm.

[0012] FIG. 2 shows Raman analyses and optical images of (a) graphene on
Si/SiO2 after removal of cells and cleaning with acetone, (b)
graphene on Si/SiO2 after removal of cells and (c) Si/SiO2
after removal of cells. Scale bars are 10 μm.

[0013] FIG. 3 shows immunostaining of cells growing on Si/SiO2, PDMS
and PET without BMP-2 growth factor. Cells are stained with DAPI (blue)
and either CD-44, MAP2, Desmin or Osteocalcin (OCN) as indicated (green).
(a-d) Cells growing on Si/SiO2, without graphene showing presence of
CD-44, and with graphene showing presence of OCN. (e-h) Cells growing on
PDMS without graphene showing some MAP2 immunostaining, and with graphene
showing staining of OCN. (i-1) Cells growing on PET without graphene
showing some staining of desmin, and with graphene showing OCN
immunostaining. Scale bars are 100 μm.

[0015] FIG. 5 shows a quantitative, functional proof of graphene-mediated
hMSCs' differentiation into osteocytes via Alizarin Red assay, (i) in the
presence (dark gray bars) or absence (black bars) of graphene, (ii) in
the absence (a) or presence (b) of additional growth factor BMP-2.
Conventional plain cover slips were used as a positive control. (c-f)
Qualitative staining via alizarin red assay of calcium deposits on PET
substrates due to osteogenesis. (c) PET without BMP-2 and without
graphene; (d) PET without BMP-2 and with graphene; (e) PET with BMP-2 and
without graphene; (f) PET with both BMP-2 and graphene. Scale bars are
100 μm.

[0016] FIG. 6 shows time-dependent immunostaining of hMSCs growing on
Si/SiO2 substrates either treated with BMP-2 or coated with
graphene. Experiments were performed from 1 hour to 15 days. (Left)
CD-44, marker for stem cells, decreased over time and completely
disappeared by Day 7. (Center) β1-integrin, marker for
cell-substrate adhesion, increased over time, reaching its peak by Day
15. (Right) OCN, marker for bone cells, became visible at Day 4 and very
intense by DAY 7. Scale bars are 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The invention pertains to methods of directing differentiation of
stem cells when cultured on graphene, in the presence of osteogenic
medium that does not require further supplementation of additional growth
factors or replenishment of growth factors. The methods and compositions
of the invention may be used for repairing or improving tissue function.
The invention is based, in part, upon data reported herein showing that
graphene provides a biocompatible scaffold that does not hamper the
proliferation of stem cells in stem cell medium and directs the stem
cells to specifically differentiate into bone cell types once cultured in
osteogenic medium.

[0018] Results showed that mono-atomic graphene coated substrates
accelerated cell differentiation to a higher extent than un-coated
substrates or cover slips. In contrast to other materials, graphene does
not require additional chemical inducers (e.g., growth factors including
BMP-2) to be continuously added or replenished to the osteogenic medium
to achieve cell differentiation. In fact, direct comparison of the
effects of graphene and growth factors on stem cell differentiation
showed that differentiation rates with graphene were comparable to the
ones achieved with common growth factors.

[0019] In one aspect, the invention pertains to a method for directing the
differentiation of stem cells into cells of interest using graphene as a
scaffold for accelerated differentiation. The term "directing
differentiation of a stem cell" as used herein is taken to mean causing a
stem cell to develop into a specific differentiated cell type. The stem
cells are grown on a graphene substrate in an appropriate culture medium
under conditions that do not require implementation with growth factors
or external stimulation, or combinations thereof. In certain embodiments
of the invention, the stem cells or progenitor cells on graphene are
grown and differentiated in vitro.

[0020] In another aspect, the invention pertains to a method for
accelerating stem cell differentiation by culturing stem cells on the
graphene substrate. The term "acceleration" as used herein means
acceleration of stem cell differentiation on graphene and in the presence
of osteogenic media as compared to differentiation only in osteogenic
media.

[0021] The results reported herein show that graphene provides unique
properties that enhance the differentiation of stem cells into cells of
interest, particularly bone cells. These differentiated cells on the
graphene substrate can be incorporated into bioimplants having improved
biocompatibility.

[0022] Graphene is a two dimensional sheet of carbon that has highly
desirable physical properties for use in tissue regeneration and medical
devices. Graphene is the strongest material known having a Young's
modulus of 0.5-1 TPa, yet it is extremely flexible and not brittle.
Graphene can be transferred onto any flat or irregular shaped surface and
graphene-coated, flexible, supporting substrates can be easily bent into
any shape required. Being only one atom thick, yet fully continuous it
also introduces the minimum amount of non-biodegradable material
preventing inflammatory or other immune responses seen with other
non-biologic materials. Graphene also serves as an impenetrable gas
barrier and can hermetically seal the substrate or implant material,
protecting it from any degradation due to external factors. As a result,
graphene may significantly strengthen bone structures or eventual
implants in addition to serving as a substrate for tissue regeneration
and/or repair.

[0023] High-quality, continuous graphene sheets can be produced on a large
scale through chemical vapor disposition on copper foil. (Bae, S., et
al., Nat. Nano, 2010, 5, 574). "Chemical vapor deposition (CVD)" refers
to a chemical process used to produce high-purity, high-performance solid
materials where substrate is exposed to one or more volatile precursors,
which react and/or decompose on the substrate surface to produce the
desired deposit. For example, graphene can be produced by exposing copper
foils to hydrogen and methane at high temperatures which react to form
single layer graphene that is deposited on the metal surface. Graphene
can be directly deposited onto any substrate, without the need to
intercalate any additional material between graphene and substrate. These
substrates include, but are not limited to, quartz, polydimethylsiloxane
(PDMS), polyethylene terephthalate (PET), and silicon wafer with 300 nm
SiO2 (Si/SiO2). In terms of biomedical applications, the
substrate of interest could consist of the metal implant or the defective
tissue itself.

[0024] Substrates that may be used for growing graphene include, but are
not limited to, copper (Cu), nickel (Ni), silicon carbide (SIC) and may
include also non-metal or non-oxide substrates. Substrates are not
limited to planar substrates but can be three dimensional forms of
nickel, copper or any other material facilitating the growth of graphene.

[0026] "Chemically modified graphene" is graphene whose structure has been
chemically altered or modified. Chemical modifications can include, but
are not limited to, covalent or ionic linking of agents to the graphene
structure or addition or substitution of substituents that may alter the
properties of graphene. Examples of agents that may be linked to the
graphene include, but are not limited to, growth factors, drugs (e.g.,
anticoagulants, such as heparin, antibiotics), antibodies, steroids,
proteins, amino acids, hormones, peptides or enzymes. Such agents can
augment of enhance the healing process or tissue repair.

[0027] "Embedded graphene" is intended to embrace any type of graphene
where a biochemical agent is incorporated into the graphene during the
coating of the substrate or thereafter. Examples of biochemical agents
that can be embedded into the graphene are those described above.

[0028] The graphene substrate useful in the present invention consists of
many micrometer ripples and wrinkles and has a high Young's modulus. The
ripples themselves provide local curvature further enhancing the
reactivity of the graphene sheets while the high Young's modulus provides
the flexibility for the out-of plane deformations which contribute to
graphene's cellular differentiation properties. As a result, the ripple
and wrinkles lead to the large scale disorder that plays a role in
protein adsorption, cell adhesion, proliferation and differentiation.

[0029] In one embodiment, graphene is multi-layer graphene. The term
"multi-layer graphene" refers to graphene that has multiple layers of
single atomic graphene on individual graphene flakes. In one non-limiting
embodiment, the graphene has ten to twenty layers. In another embodiment,
the graphene has five to ten layers. In yet another embodiment, the
graphene has one to five layers. In another embodiment of the invention,
the graphene is single layer graphene. As used herein, the term "single
layer graphene" refers to a graphene monoatomic sheet that has less than
or about 5% two or three layer graphene. For example, graphene grown on
copper is self terminating producing single layer graphene that has less
than 5% two and three layer graphene flakes. In one non-limiting
embodiment, the graphene has about 5% two and three layer graphene. In
another embodiment, graphene has less than 5% two and three layer
graphene.

[0030] According to the invention, a stem cell is cultured in the presence
of graphene. In one embodiment, the graphene is in direct contact with
the cells. In another embodiment, the graphene is in contact with the
culture media, and in direct contact with the cells. For example, stem
cells are seeded on graphene coated substrate and then placed in culture
media.

[0031] A variety of stem cells of various types and stages of
differentiation can be used in the invention and include but are not
limited to, for example, totipotent, pluripotent, multipotent and
unipotent stem cells. In one embodiment of the invention, the stem cell
is an embryonic stem (ES) cell. In another embodiment of the invention,
the stem cell is a progenitor stem cell. In yet another embodiment, the
stem cell is a mesenchymal stem cell.

[0032] Of particular interest are mesenchymal stem cells (MSCs) which can
differentiate in vitro, in a variety of connective tissues or progenitor
cells, including, but not limited to, mesodermal (osteoblasts,
chondrocytes, tenocytes, myocytes and adipocytes), ectodermal (neurons,
astrocytes) and endodermal (hepatocytes) derived lineages. The term
"mesenchymal stem cell" and "marrow stromal cell" are often used
interchangeably, so it is important to note that MSCs encompass
multipotent cells from sources other than marrow, including but not
limited to, muscle, dental pulp, cartilage, synovium, synovial fluid,
tendons, hepatic tissues, adipose tissue, umbilical cord, and blood,
including cord blood.

[0033] While stem cells exemplified herein are differentiated into bone
cells, differentiation into any desired "cell of interest" is
contemplated. Examples include, but are not limited to, osteocytes,
chondrocytes, adipocytes, muscles cells, nerve cells and cardiac
myocytes. In one embodiment, the differentiated cell is a chondrocyte. In
another embodiment, the differentiated cell is an osteocyte. In another
embodiment, the differentiated cell is a cardiac myocytes. In a further
embodiment, the differentiated cell is a muscle cell. In yet another
embodiment, the differentiated cell is a nerve cell. In another
embodiment, the differentiated cell is an osteoblast. In another
embodiment, the differentiated cell is an adipocyte. In another
embodiment, the differentiated cell is a hepatocyte.

[0034] The invention also applies to a variety of stem cells of various
types and stages of differentiation, and cultured in media that promotes
differentiation toward a particular type of cell. The term "culture
media" as used herein means any liquid or solid preparation made
specifically for the growth, storage or transport of microorganisms or
other types of cells. The variety of media that exist allow for the
culturing of specific organisms and cell types, such as differential
media, selective media, test media and defined media. In one embodiment,
the culture medium is chondrogenic. In another embodiment the culture
medium is osteogenic. In another embodiment, the culture medium is
myogenic. In another embodiment, the culture medium is neurogenic. In
another embodiment, the culture medium is adipogenic. In another
embodiment, the culture medium is hepatogenic. For example, human
mesenchymal stem cells (hMSCs) can be placed on graphene in osteogenic
media to obtain osteogenic differentiation.

[0035] Conventional osteogenic medium contains dexamethasone, which can
lead to osteogenic differentiation. However, it is usually administered
in combination with other agents, growth factors or external stimulants
to achieve differentiation through a synergistic effect since
differentiation in osteogenic medium occurs over prolonged periods of
time. "Growth factors" include naturally occurring substances capable of
stimulating cellular growth, proliferation and cellular differentiation.
For example, bone morphogenetic protein-2 (BMP-2) is a growth factor that
plays an important role in the differentiation of cells into bone and
cartilage. As used herein, "external factors" or "external stimulants"
are external sources of mechanical, acoustic or electromagnetic energy
that can stimulate cellular proliferation and differentiation. For
example, radiowaves or electromagnetic radiation can be used to supply
cells with the sufficient energy needed to promote cellular growth.

[0036] According to the invention, the graphene can be employed not only
in tissue culture, but wherever it is desired to stimulate growth and/or
repair of bone, cartilage, muscle, or nervous tissue in a host. The stem
cells can be cells already present at a particular location, or
implanted, or injected. In one embodiment, stem cells are stimulated on
graphene in vitro. In a further embodiment, progenitor cells are
stimulated directly using graphene. In certain embodiments, the stem
cells seeded on graphene are implanted as part of a tissue or prosthesis
or treatment of structures so destined for insertion or implantation into
a host.

[0037] One example of such a structure is a matrix for bone or cartilage
growth or regeneration. Examples include, but are not limited to a
demineralized bone matrix (e.g., composed primarily of collagen and
non-collagenous proteins), devitalized cartilage matrix, or other
artificial matrix for bone or cartilage repair. Other porous scaffolds
(ceramics, metals, polymers and nano-reinforced) are osteoconductive and
promote bone ingrowth, with osteoinductive properties provided by
incorporation of peptides, hydroxyapetite and cytokines known to
influence bone cells.

[0038] In one embodiment, collagen, particularly collagen type II, is used
to promote chondrogenic differentiation of stem cells on graphene. In
another embodiment, osteogenic matrix is used to promote osteogenic
differentiation of stem cells on graphene. In another embodiment,
graphene seeded with stem cells is implanted at the regeneration site. In
another embodiment, stem cells on graphene are incorporated into an
implant or prosthesis. In yet another embodiment, progenitor cells on
graphene are incorporated into an implant or prosthesis. In another
embodiment, the implant is coated with graphene and osteogenic
differentiation is promoted at the implant site. In another embodiment,
an implant made of TiO2 or any other medical implant material, is
coated with graphene and osteogenic differentiation are promoted at the
implant site. In another embodiment, the graphene is grown on the implant
and differentiation is promoted at the implant site. In another
embodiment, the graphene is a three-dimensional scaffold serving as
implant material and differentiation is promoted by the graphene implant.
In another embodiment, graphene can be used as bonefilling material.
Medical implant materials include, but are not limited to, graphene,
metal, metal alloy (e.g., stainless steel or Cobalt Chrome), metal oxide
(e.g., TiO2), oxide, ceramic, composite materials and plastics.

[0039] Preferably, graphene would be directly implanted at the site of
defective tissue, to provide mechanical support while promoting stem
cells growth and proliferation in a particular cell lineage. Graphene
offers the potential to be further functionalized and/or embedded with
biochemical agents to enhance healing process and tissue repair. Also,
graphene can be used as a temporary scaffold to direct cell
differentiation into a specific lineage, after which, it could be
separated from the differentiated cells and completely discharged.

[0040] Thus, the matrices can include bone- or cartilage-specific matrix
components and are populated with bone or cartilage progenitor cells,
which are stimulated according to the invention.

[0041] The invention also provides for a composition for stimulating
and/or differentiating stem cells or progenitor cells. The compositions
are suitable for cell growth and comprise stems cells on a graphene
substrate exposed to culture media. In one embodiment, the composition
comprises graphene coated or placed on a biocompatible material. In
another embodiment, the composition comprises stem cells on a graphene
coated biocompatible material. Biocompatible materials can include
natural or synthetic materials used to replace part of a living system
(e.g., tissue or organ replacement) or to function in intimate contact
with living tissue.

[0042] The method of the invention is also applied to the manufacture and
use of medical implants, such as an orthopedic or a dental implant. The
implant can be a metal implant, such as an artificial hip, knee, or
shoulder, to which the bone must meld. Other examples include dental
implants. The implants are prepared with graphene attached surfaces that
are to be fused to bone, providing an improved surface that enhances
growth of bone forming cells. The implant can also be made of a composite
material such as a fiber composite. For example, orthopedic implants can
be made from composite material strengthened by the addition of graphene.
The implants can be implanted directly, or incubated with osteoblasts
from the recipient prior to implantation.

[0043] When implanted or injected, stem cell development is often governed
by the site of implantation or the site in the body to which the stem
cell is home. According to the invention, differentiation of stem cells
and progenitor cells can also be directed in vitro by selection of media
components and/or matrix components. For example, cytokines, and growth
factors that promote osteogenic differentiation include various isoforms
of bone morphogenetic protein (BMP) such as BMP-2, -6, and -9,
interleukin-6 (IL-6), growth hormone and others. (See, e.g., Heng et al.,
2004, J. Bone Min. Res. 19, 1379-94). Cytokines and growth factors that
promote chondrogenesis include various isoforms of TGF-β and bone
morphogenetic protein, activin, FGF and other members of the TGF-β
superfamily. Osteogenesis of chondrogenesis is favored by naturally
occurring or synthetic cartilage extracellular matrix (ECM) material. For
example chondrogenesis is favored by naturally occurring or synthetic
ECM. Such an ECM can comprise collagenous proteins such as collagen type
II, proteoglycans such as aggrecan, other proteins and hyaluronin. (See,
e.g., Heng et al., 2004, Stem Cells 22, 1152-67). Phenotypic markers
expressed by cells of the various lineage are well known in the art.

[0044] The invention further provides kits for differentiating stem cells.
The kits comprise graphene for controlled and accelerated differentiation
of the stem cells. The graphene can be provided separately from the stem
cells or coated on the containers used for culturing stems cells. In
another embodiment, the kit contains graphene incorporated onto a
support, such as a scaffold on or within which stem cells or progenitor
cells are stimulated and/or differentiated. In a further embodiment, the
kits contain instructions on how to use the invention to obtain
stimulated or differentiated cells using graphene and the appropriate
culture media. Optionally, the kits further contain media formulations
selected to promote differentiation to osteocytes, chondrocytes, or other
differentiated cell types. Suitable media include, but are not limited
to, adipogenic media, osteogenic media, chondrogenic media, myogenic
media, neurogenic media, hepatogenic media

[0045] In one embodiment of the invention, the differentiated stem cells
are used to identify and/or isolate biological compounds, including but
not limited to proteins and nucleic acids characteristic of the
stimulated or differentiated state of the cells. Such biological
compounds are useful for example, as markers of differentiation and as
targets for antibodies and other agents. Fluorescent antibodies, specific
for immunostaining of typical proteins produced by defined cell lines,
can be used to confirm whether differentiation has occurred or not. A few
examples are the fluorescent antibody for CD-44 (which is typical of
MSCs), or DESMIN (D-33, specific for muscle cells), or antibody for MAP-2
(used as a marker for neurons) or OCN (specific for osteocytes) or
β1-integrin (protein produced when cells have increased
adhesion to the substrate underneath). As an example, hMSCs incubated in
osteogenic media for 14 days, show the ability to bind OCN only in the
presence of graphene-coated substrates, while they immunostain for CD-44
on cover slips or uncoated substrates.

EXPERIMENTAL PROCEDURES

Substrate Preparation

[0046] Graphene was grown on copper foils by chemical vapor deposition at
1000° C. in a mixture of hydrogen and methane as discussed
elsewhere. (Li, X., et al., Science 2009, 324, 1312). The graphene film
was mechanically supported by a thin film of polymethyl methacrylate
(PMMA) (Microchem) and the copper foil was etched in a weak solution of
ammonium persulfate (Sigma). The graphene coated with PMMA was
transferred to deionized water to remove residues and the transfer was
completed by gently contacting graphene with the substrate and lifting it
out of the water. To avoid any residues from the transfer process the
samples were left in warm acetone for 12 hours followed by 3 hours in
isopropanol. In a final step the Si/SiO2 substrates were annealed in
Ar/H2 90/10 wt % for 7 hours at 300° C. to further reduce
impurities in the graphene layer. However, note that Si/SiO2 without
the additional step of annealing showed the same cell viability and
induced stem cell differentiation at the same rate (data not shown).

[0047] Large-scale graphene used in this study was synthesized by the
chemical vapor deposition method on copper foils. After growth, copper
was etched and the same batch of graphene was transferred to four
distinct substrates used in this study according to methods discussed
elsewhere. (Li, X., et al., Science 2009, 324, 1312) The influence of
graphene on stem cell growth was studied by investigating four distinct
substrates with widely varying stiffness and surface roughness: (1)
polydimethylsiloxane (PDMS), (2) polyethylene terephthalate (PET), (3)
glass slide and (4) silicon wafer with 300 nm SiO2 (Si/SiO2).
Plain cover slips without graphene were used as a control or reference
for normalization. Atomic Force Microscopy (AFM) was used to analyze the
surface roughness of the various substrates with and without graphene
coating.

[0048] Transferred to PET, PDMS, and Si/SiO2, the graphene sheet
exhibits nano-ripples with high density compared to graphene on glass
slide. Despite being only one atom thick, on Si/SiO2 substrates with
well-defined oxide thickness, graphene can be easily seen with a simple
conventional optical microscope. First cell viability was studied with
cells cultured in normal stem cell medium. Next, stem cell
differentiation was examined in cells cultured on conventional osteogenic
media.

Cell Viability and Morphology Mesenchymal Cells in the Presence of
Graphene.

[0050] hMSCs Differentiation into Osteogenic Lineage.

[0051] hMSCs (20,000 cells/well (24 well plate)) were seeded on uncoated
(control) and graphene coated (test) chips and cultured in normal stem
cell medium. Post confluence (2 weeks), cells growing on each chip were
transferred to new well plate and washed 3 times with 2 ml of PBS. 1 ml
of PBS was added to each well followed by 5 μl of 1 mM Calcein
acetoxymethyl ester (Calcein AM) and incubated at room temperature for 15
minutes. After removing the unbound stains, the chips were inverted on to
glass slides mounted with vectashield with 4,6'diamidino-2-phenylindole
(DAPI) (H 1200, Vector labs) and visualized under fluorescence microscope
(Nikon AZ-100 multipurpose microscope). Pictures were taken at 4
different positions of the chips and processed by image J software to
count the number of viable cells to the number of nucleus as determined
by staining with DAPI. Cell viability was measured by comparing the cell
numbers for each substrate with the cells counted on cover slips. In
addition, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assays were carried out, in which cytotoxicity evaluation was based
on the activity of enzymes to reduce MTT to formazan dyes, giving a
purple colour. (Mosmann, T., J. Immun. Met. 1983, 65, 55). Experiments
were carried out in triplicates, following the procedure reported in
supporting document. The morphology of the hMSCs on different substrates
was compared according to the image as seen in the form of calcein AM
staining (FIG. 1).

[0052] Cell cytotoxicity of graphene was tested by comparing cell counts
for all four substrates with and without graphene coverage and found that
graphene does not hamper stem cells' normal growth over the whole period
of investigation (1-18 days). On the contrary, MTT assay showed that
cells grew better on graphene covered substrates in particular on the
softest substrates, i.e. PDMS and PET. From FIG. 1 (a) it can be seen
that, independent of the substrate, there is no significant difference
(p>0.05) in cell viability between graphene-coated and uncoated
substrates. MTT assays were also performed to confirm the cell viability
data. Again, regardless of the substrate, there was no difference
(p>0.05) between uncoated and graphene-coated substrates,
demonstrating that cell growth was indeed not affected by the presence of
graphene. Note that cell viability is lower on PET and PDMS independent
of the presence of graphene.

[0053] A similar conclusion can be reached by just comparing cell
morphology with and without graphene (FIG. 1 (b-i)). In general, the
presence of graphene did not influence the shape of the cells in
comparison to uncoated substrates. Mesenchymal stem cells maintained
their spindle-shape across glass slides and Si/SiO2 after 15 days of
incubation. Here stem cells presented the usual elongated structure with
noticeable filopodia extensions and cellular propagation fronts. In the
case of PET and PDMS, cells showed rounded or irregular morphology, most
probably due to poor adhesion to the substrate. This suggests that
graphene does not hamper the normal growth of stem cells and that the
incorporation of this material in implants or injured tissues would not
affect the physiological conditions of the microenvironment. In fact,
Raman measurements and visual inspection of the samples after cell
incubation and subsequent removal clearly showed that the graphene sheets
remained largely intact.

[0054] Raman spectra of graphene on Si/SiO2 after cell removal and
subtraction of the background, clearly show the G and 2D peaks, which
represent the Raman "fingerprints of graphene" (FIG. 2). Note also, that
the absence of the D-peak at 1350 cm-1 indicates the lack of defects
to the graphene crystal lattice (Ferrari et al. PRL, 2006, 97, 187401).
The optical images also clearly shows that the graphene sheet remains
largely intact.

Immunofluorescence of hMSCs

[0055] hMSCs at 20,000 cells/well (24 well plate) were seeded,
osteoinduced and incubated up to confluence (2 weeks) as reported above.
The cells on all the chips were fixed by treating them with ice cold
50%/50% methanol/acetone. After 5 minutes, methanol/acetone was removed
and the chips were left open inside the laminar hood to be air dried.
After the chips were completely dried, the fixed cells were treated with
10% FBS (blocking agent) in PBS for 20 minutes. The blocking agent was
aspirated out and 5 μl of different antibodies to cellular markers
(CD-44 for hMSCs, OCN for osteoblasts, Desmin for muscle cells and MAP2
for neuronal cells) were added on to separate chips (previously marked).
After 1 hour the cells on the chips were extensively washed in Millie
water for 5 minutes and then rinsed in PBS 1× for 5 minutes. After
that, 100 μl of diluted (1/100) FITC-goat antimouse antibody were
added on to each chip and incubated at room temperature. After 30 minutes
the cells were washed with MilliQ® water for 5 minutes and then rinsed
in PBS 1× for 5 minutes. The chips were inverted on to glass slides
mounted with vectashield with DAPI (H 1200, Vector labs) and visualized
under fluorescence microscope (Nikon AZ-100 multipurpose microscope).

[0056] Osteogenic differentiation was evaluated over a time frame of two
weeks. Uncoated substrates were subjected to BMP-2 (75 ng/mL, added every
3 days) and compared to graphene coated substrates at 1 hour and at Day
1, 4, 7, 10 and 15 in terms of binding to CD-44 (which stains hMSCs),
β1-integrin (which indicates cell-substrate adhesion) and OCN
(which indicates bone cells). The above mentioned procedure was followed
for the immunofluorescence and imaging purposes.

[0057] Next, specific markers were used to determine the conversion of
hMSCs into specific cell types when cultured in osteogenic media. Note
that conventional osteogenic medium does contain dexamethasone, which can
lead to osteogenic differentiation by itself. However, it is usually
administered in combination with other agents and growth factors such as
BMP-2 to achieve differentiation through a synergistic effect. In none of
the un-coated substrates studied here, the osteogenic medium alone was
sufficient to lead to osteogenic differentiation over the whole duration
of the experiment (15 days). In the absence of graphene, stem cells on
cover slips, on glass slides and on Si/SiO2 did not differentiate:
this was demonstrated by immunofluorescent staining of two typical
protein markers, namely CD-44 for hMSCs and osteocalcin (OCN) for
osteoblasts (FIG. 3). These three substrates showed a CD-44-positive
staining and the absence of OCN. However, once these stiff substrates
were coated with graphene, hMSCs lost their ability to bind the
fluorescent antibody specific for CD-44 expression, suggesting they
underwent a different fate. In fact, hMSCs immunostained for OCN,
indicating osteogenic differentiation. On uncoated PDMS, hMSCs did not
stain CD-44 but they showed a weak expression of MAP2 (typical neuronal
marker). On the other hand, in the case of uncoated PET, desmin (D33, a
muscle cell marker) staining but not CD-44 was observed. However, once
coated with graphene, hMSCs growing also on these softer substrates bound
specifically to OCN only, demonstrating that graphene is the driving
force of bone cell formation, regardless of the underlying substrate.

[0058] This is most clearly seen in the immunofluorescent staining of
cells on a Si/SiO2 wafer, which are cultured in osteogenic medium
but only partially covered by graphene. Despite the stiffness of the
substrate, specific immunostaining for OCN was only observed in the area
covered by graphene. The boundary separating the graphene coated region
from the uncoated region is clearly visible even from the
immunofluorescent image (FIG. 4).

Alizarin Red Staining and Quantification

[0059] hMSCs (20,000 cells/well (24 well plate)) were seeded in to the
control and the test well plate. After 24 hours, osteogenesis was induced
by replacing the original medium with osteogenic medium, which was
changed every 3 days up to confluence (2 weeks).

[0060] Alizarin red staining was performed using the protocol adapted from
Chemicon Mesenchymal Stem cell Osteogeneis kit Cat. No. SCR028. Briefly,
the medium was aspirated out from each well and cells were fixed with ice
cold 70% ethanol for 1 hour at room temperature. Then the cells were
rinsed twice with MilliQ® water followed by addition of 2 ml of
alizarin red (Sigma) solution for each well and incubated for 30 minutes.
Finally the unstained alizarin red was washed with MilliQ® water and
the chips were visualized under microscope (Nikon eclipse TE2000-U,
Japan). Cells with calcium deposits due to bone nodule formation were
stained red. Alizarin red quantification was done using a previously
reported procedure. (Tataria, M., et al., J. Pediatr. Surg. 2006, 41,
624).

[0061] Alizarin Red assay is used to assess the presence or absence of
calcium deposits due to bone nodule formation.

[0062] The extent of calcium deposition on each substrate was compared
using the alizarin red staining results, with and without graphene
coating, in the absence of the typical growth factor BMP-2 (FIG. 5). A
strong increase in calcium deposit with graphene coating is observed for
all substrates. While the effect is more pronounced with the stiffer
substrates, surprisingly graphene had a similar effect also on the softer
substrates PET and PDMS. It should be noted that in the absence of growth
factors both PDMS and PET are known to be less favorable towards
osteoblasts. (Konttinen, Y. T., et al., Clin. Orthop. Relat. Res. 2005,
430, 28). Yet the presence of graphene induced a drastic change of their
natural behavior similar to what has been observed with apatite coating
on such polymers. (Kawai, T., et al., Biomat. 2004, 25, 4529; Kim, H.-M.,
et al., J. Mat. Sci. Mater. Med. 2000, 11, 421; Kim, H.-M., et al., J.
Biomed. Mater. Res. 1999, 46, 228). Osteogenic medium alone was not
sufficient to induce differentiation: within the 15 day time frame of the
experiment, the control represented by cover slips in osteogenic medium
without graphene, i.e. hMSC cultured on ordinary tissue culture plate,
did not show any calcium deposition.

[0063] The impact of graphene on softer substrates such as PET became even
more evident in a parallel study, where graphene's influence to that of
BMP-2 was directly compared after 15 days of incubation (FIG. 5). In the
absence of both graphene and BMP-2, no bone nodule formation was observed
as indicated by negative alizarin red staining. As expected, positive
staining with identical samples after the addition of BMP-2 was observed.
On the other hand graphene-coated PET showed a positive staining even
without BMP-2. Experiments were also performed where both graphene
coating and BMP-2 treatment were combined. In the case of PET and PDMS,
significant enhancement of calcium deposits were observed compared to the
above-mentioned samples, which were either only coated with graphene or
only treated with BMP-2. This enhancement was specific to soft
substrates, and much less evident on the stiffer glass slides and
Si/SiO2.

FACS Analysis (Flow Cytometry Experiments)

[0064] The hMSCs grown on different substrates (i.e. cover slips,
uncoated-Si/SiO2 and graphene-coated Si/SiO2) were subjected to
differentiation with osteogenic medium (in the presence or absence of
BMP-2) and analyzed after 14 days by fluorescent-activated cell sorting
(FACS). The harvested cells were fixed with 4% paraformaldehyde by
incubating for 20 minutes. After centrifugation at 1500 RPM for 5 minutes
and washing with PBS, the cell pellets were suspended in 100 mM glycine
for 10 minutes to quench. The cells were then again centrifuged and
washed with PBS and permeabilized by incubating in 50 μl of 0.1%
Triton X for 30 minutes. Subsequently, the cells were washed with PBS and
were incubated with mouse antihuman osteocalcin antibody for 30 minutes
at room temperature. The cells were further washed with PBS and incubated
with FITC conjugated goat anti mouse IgG for another 30 minutes. Finally
the cells were washed 2-3 times with PBS and were analyzed using BD LSR
II flow cytometer (Becton Dickinson).

[0065] FACS histogram confirmed negligible osteocalcin positive cells in
case of hMSCs on substrates incubated in normal medium. The expression of
osteocalcin was maximal for all the substrates in osteogenic media with
both graphene and BMP-2. This is similar to the results obtained with the
alizarin red quantification and confirms the synergistic effect when both
graphene and BMP-2 were concurrently present. Interestingly, osteogenic
medium with graphene, but in the absence of BMP-2, reached almost the
same levels of cell differentiation (83%) as those in osteogenic medium
with both graphene and BMP-2 (100%).

Time Dependence of Differentiation.

[0066] An important parameter for practical applications is also the time
a material takes to induce bone cell differentiation. To that purpose a
study was conducted to see how fast cells on graphene-coated Si/SiO2
substrates differentiate over a time frame of 15 days in comparison to
cells growing on uncoated Si/SiO2, but treated with BMP-2 (FIG. 6).
These samples were studied at specific time points of 1 hour and 4, 7, 10
and 15 days. Interestingly, both BMP-2-treated and graphene coated
substrates were able to induce cell differentiation at the same rate.
More precisely, hMSCs on neither substrate showed any sign of osteoblast
formation until Day 4. This is demonstrated by the intensity of
fluorescence due to CD-44 marker, which is characteristic for stem cells
and clearly visible already after one hour of incubation. Conversely,
fluorescence due to CD-44 decreased remarkably by DAY 4 and completely
disappeared by DAY 7. On the other hand, a progressive enhancement of
fluorescence was observed due to OCN (indication of terminal osteogenic
differentiation) and β1-integrin, a protein indicating
cell-substrate interaction. The results confirmed a successful
differentiation into bone cells with a strong adhesion to the substrates
by DAY 7 for both types of samples. Si/SiO2 substrates treated with
a) only BMP-2 and b) only graphene were able to accelerate cell
differentiation at the same rate over a period of 15 days of incubation.
Equally important, in contrast to graphene, BMP-2 needed to be
administered every three days during the course of the experiment due to
the very short half-lives of BMP-2 again showing graphene as a worthy
replacement of biochemical growth factors. (Balmayor, E. R., et al.,
Clin. Orthop. Relat. Res. 2009, 467, 3138; Dragoo, J. L., et al., J.
Orthop. Res. 2003, 21, 622).

Control Experiments.

[0067] To confirm that graphene is critical for the observed stem cell
differentiation, control experiments were performed with both amorphous
carbon thin films and highly oriented pyrolytic graphite (HOPG) samples.
Following identical experimental protocols, it was observed that while
both types of samples did support cell proliferation, none of them led to
cell differentiation.

[0068] Cells were cultured on graphene or HOPG in osteogenic medium. After
4 days the fluorescence deriving from the antibody specific for CD-44
expression was significantly lower for cells grown on graphene than for
cells on HOPG. At the same time, specific immunostaining for OCN was
already detectable with cells grown on graphene, while only the DAPI
stained nuclei were visible for cells on HOPG.

[0069] The observed effect is almost certainly due to a complex interplay
of mechanical, chemical and electrical properties of graphene and the
interactions between graphene and cells, as well as graphene and
supporting substrates. The disparities between the results obtained with
graphene and HOPG point towards mechanical properties and surface
morphology as the decisive factors. AFM images of graphene and HOPG
clearly show the difference in their topography. While locally
(˜1001 nm) the two systems have comparable surface morphology, on a
larger scale they look very different. CVD graphene consists of many
ripples and wrinkles on the micron scale. Such localized out-of plane
deformations are completely absent in HOPG graphite, the surface of which
consists instead of a large number of micron size terraces.

[0070] The fact that (HOPG) graphite is made out of weakly bound graphene
planes may be equally important. In the presence of lateral forces such
materials easily shear off and are therefore, commonly used in
lubricants. In the specific context of cell adhesion and in view of the
(lateral) contractual forces cells exert on the surface, this effect may
hamper strong cell adhesion. Note that cells can mechanically "sense"
lower lying layers down to several tens of micrometers. In the case of
graphene, the cells sense the underlying (amorphous) substrate instead.

CONCLUSIONS

[0071] To summarize, the presence of graphene did not influence the shape
and the growth of the cells in normal stem cell media, demonstrating
biocompatibility and suggesting that the incorporation of this material
in implants or injured tissues would not affect the physiological
conditions of the microenvironment. In the presence of an osteogenic
medium, graphene-coating helped by remarkably accelerating the
differentiation of hMSCs at a rate comparable to differentiation under
the influence of BMP-2. This represents a critical aspect to its
successful use for stem cell-based regenerative medicine strategies. In
contrast to other substrates, graphene is neither brittle nor does
require further nanoscale patterning or functionalization. In addition it
is scalable and provides a cost effective way to prepare scaffolds for
biological tissues. Currently graphene is only available in form of
sheets and we envision a promising role of graphene located between
implants and the surrounding tissues. However, the conditions under which
graphene is grown are being constantly improved. There is for example a
strong effort in establishing graphene growth at much lower temperatures.
Thus, growth on alternative biocompatible and biodegradable surfaces,
potentially even without the need to resort to catalytic metal films,
seems feasible. Even the growth on 3D scaffolds has recently been
demonstrated. (Chen, Z., et al., Nat. Mater., 2011,
doi:10.1038/nmat3001).

[0072] The teachings of all patents, published applications and references
cited herein are incorporated by reference in their entirety.

[0073] While this invention has been particularly shown and described with
references to example embodiments thereof, it will be understood by those
skilled in the art that various changes in form and details may be made
therein without departing from the scope of the invention encompassed by
the appended claims.